Protein-tyrosine phosphatases (PTPases), in particular the non-receptor type PTP1B and the receptor-type PTPase LAR, have important roles in balancing the steady-state tyrosine phosphorylation of the insulin receptor (IR) and its substrate (IRS) proteins. The long-term objectives of this project are to fully characterize the mechanism of action of PTP1B and LAR and determine how they are regulated in normal physiology and in insulin-resistant disease states. In the next period of support, the investigators propose to study the following: Specific Aim 1 is a major new initiative to test the novel hypothesis that the reactivity of the catalytic cysteine thiol of PTP1B and LAR is modulated by reversible oxidation and conjugation in situ, as has recently been shown for other growth factor signaling systems. Using an anaerobic chamber, we will evaluate the activation state and the biochemical structure of PTP1B and LAR as isolated from a variety of insulin-sensitive cell types following exposure to insulin or H202, and in states of altered oxidative stress, including the hyperglycemia of diabetes. Specific Aim 2 will continue our established studies on the biosynthesis, processing and compartmentalization of PTP1B and LAR in insulin-sensitive cell types to test the hypothesis that the proteolytic cleavage, shedding of the LAR ectodomain, internalization of LAR and subcellular redistribution of PTP1B plays an essential role in their regulation of the insulin action pathway. The domains of physical interaction between LAR and the IR will be identified and the thiol reactivity of LAR and PTP1B will be evaluated in different subcellular fractions. Whether PTP1B and LAR have additive roles in the regulation of the IR and IRS proteins in insulin signaling will be evaluated. Specific Aim 3 will expand our finding that IRS-1, PTP1B and GRB2 form a ternary complex in vitro that enhances the dephosphorylation of IRS-1 by PTP1B. We will test the hypothesis that this complex forms in vivo with functional significance in the regulation of insulin signal transduction. In parallel studies, the investigators will evaluate the functional role of the adapter protein CrkII, which also binds PTP1B and IRS-1 and also test how protein complex formation might regulate the dephosphorylation of IRS-2. New data from each of these aims will provide insight into the regulation of insulin action and may indicate novel targets for enhancing insulin sensitivity in common insulin-resistant states such as type 2 diabetes mellitus and obesity.